title: 3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent.
abstract: A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [125I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes from Halobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[14C]-cyanate binding to BR. [125I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generated in situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.
authors.affiliation: Institute of Biochemistry, University of Berne, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Dolder
authors.forename: H
authors.initials: H
authors.lastname: Michel
authors.forename: H
authors.initials: H
authors.lastname: Sigrist
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D000838:Anions; D001389:Azirines / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D006217:Halobacterium / Q000032:analysis; D006801:Humans; D006838:Hydrocarbons; D017879:Isothiocyanates; D008565:Membrane Proteins / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives; D010446:Peptide Fragments / Q000378:metabolism; D010782:Photolysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D013861:Thiocyanates / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D001389:Azirines; D003432:Cross-Linking Reagents; D006838:Hydrocarbons; D017879:Isothiocyanates; D008565:Membrane Proteins; D010446:Peptide Fragments; D013861:Thiocyanates; C005441:phenylisothiocyanate; C030011:carbene; D001436:Bacteriorhodopsins; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013820:Thermolysin; D008697:Methane